Final Practical

Label 4 tubes and then zero spectrophotometer with DI water (405 nm), add Amylase reagent to each tube and incubate at 37 C for 3 minutes. Add appropriate sample to each tube one at a time and then read immediately. Read absorbance at 30 seconds intervals for 2 minutes. Determine the mean absorbance difference per minute, and multiply that by 4824 to get result in U/L.

Identify interfering substances for amylase determination

Drugs and substances

reference range for amylase serum

25-125 U/L

reference range for amylase urine

1-17 U/hour

principle of the amylase determination

The principle of this method is based on alpha-amylase enzyme which catalyzes the hydrolytic cleavage of starch and glycogen. The procedure is based on the Wallenfels modification which uses substrate p-Nitrophenyl-D-maltoheptaoside (PNPG7) with the terminal glucose blocked to reduce spontaneous degradation of the substrate by glucosidase and glycoamylase. Amylase hydrolyzes p-Nitrophenyl D-maltoheptaoside (PNPG7) to p-Nitrophenyl-maltotriose (PNPG3) and maltotetraose. Glucoamylase hydrolyzes PNPG3 to p-Nitrophenylglycoside (PNPG1) and glucose. Then PNPG1 is hydrolyzed by glucosidase to glucose and p-Nitrophenol, which produces a yellow color. The rate of increase in absorbance is measured at 405 nm and is proportional to the amylase activity in the sample.

concentration of amylase using change in absorbance over time.

U/L = ?Abs./min. x 4824

State classic reference method for amylase activity

Wallenfels method

Briefly describe the classic reference method for amylase activity

Wallenfrels method uses PNPG7 which is hydrolyzed by amylase in the specimen to PNPG3 and maltotetraose. PNPG3 is then is hydrolyzed by glucoamylase to PNPG1 and glucose. PNPG1 is hydrolyzed by glucosidase to glucose and p-Nitrophenol which produces a yellow color. The rate of increase in absorbance is proportional to the amylase activity in the sample.

Briefly describe the analytical methods which may be used to determine amylase isoenzymes.